The accurate detection of glucose is critical for clinical diagnostics and metabolic monitoring. This study focused on the successful synthesis and incorporation of the glucose dehydrogenase (GDH) gene sequence from Burkholderia cepacia into a plasmid, followed by codon optimization and modification of cysteine motifs for increased expression in E. coli. The engineered E. coli strain demonstrated efficient heterologous expression of stable GDH. Remarkably, the recombinant strain not only enabled cost-effective GDH production but also exhibited excellent glucose catalytic activity, with a detection range of 1 μM to 10 mM and a minimum detectable concentration of 1 μM. This functionality makes it suitable for applications of AuNPs/SPE/glucose biosensors, offering high sensitivity for glucose detection—a novel achievement not previously reported. These findings have the potential to advance the clinical utilization of AuNPs/tetracysteine/GDH-modified biosensors and may promote the development of GDH-based electrochemical biosensors.